Engineering archaeal DNA polymerases for biotechnology applications

• Main Author: Elshawadfy, Ashraf Mohamed
• Published: University of Newcastle Upon Tyne 2012
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606814

The DNA polymerase from the archaeon Pyrococcus furiosus (Pfu- Pol) is commonly used in the polymerase chain reaction (PCR). The enzyme has high thermostability and is very accurate due to the presence of 3'---5'exonuclease (proofreading) activity. Unfortunately, the polymerase has relatively low processivity, limiting its ability to amplify long stretches of DNA relatively quickly. In this project, two approaches have been used in an attempt to improve the performance and processivity of Pfu DNA polymerase in PCR applications. In the first, the overall positive charge of the protein has been increased; predicted to increase electrostatic interactions between the negatively charged DNA and the more positively charged proteins. In the second, we have prepared a set of Pfu-Pol mutants in an attempt to make Pfu-Pol more similar to KODl; a polymerase isolated from a related hyperthermophilic archaeon Thermocococcus kodakaraensis. KODl is known to have a higher processivity than Pfu-Pol and both share a 3'---5' proof reading exonuclease activity. A PCR-based protocol was used to introduce the desired mutations.